PROJECT SUMMARY The long-term goal of this research program is to understand how human cells respond to replication stress, the slowing or stalling of DNA replication induced by barriers to replication fork progression. Fork stalling barriers can be induced environmentally by alkylating agents, crosslinking agents and aldehydes, or they can arise from endogenous DNA damage, secondary DNA structures or interference with transcription. Prolonged replication fork stalling can lead to DNA break formation and a failure to complete DNA replication. This is turn can result in genome instability and mutagenesis, properties that drive cancers and affect their sensitivity to chemotherapy. Fortunately, cells can respond to replication stress in multiple ways, depending on the barrier encountered. Two crucial components of the replication stress response involve fork slowing and replication fork reversal, a process that involves reannealing of the unwound template DNA and annealing of nascent DNA strands. Fork reversal is thought to promote DNA repair, stabilize the replication fork and facilitate certain forms of fork restart. It can also allow for the error-free replication of damaged DNA by providing the undamaged sister chromatid as a template for DNA replication. When fork reversal occurs and how it is regulated in the cell are poorly understood. In the previous funding period, we made the surprising finding that the DNA translocase and fork reversal enzyme, HLTF, is needed to slow replication forks during replication stress. This fork slowing requires HLTF's fork reversal activity. HLTF-deficient cells also proliferate better under replication stress conditions. This combination of unusual phenotypes is unique to HLTF among fork remodelers. The objective of this application is to characterize this stress-resistant, potentially error-prone replication mode at the molecular and cellular level, thereby addressing fundamental questions about the balance of replication fork progression and fork reversal during replication stress. In the first aim, we will explore how HLTF processes replication forks to slow their progression and induce DNA breaks. In the second aim, we will determine the mechanisms and mutagenic consequences of stress-resistant DNA replication and unrestrained fork progression. Finally, in the third aim, we will probe the mechanisms of cell survival under replication stress, searching for targets that contribute to stress resistance in HLTF-deficient cells. These experiments will capitalize on cutting-edge genomic and proteomic approaches to solve fundamental problems regarding replication fork reversal and replication stress resistance. !